Supplementary Material for: Upregulation of RASSF1A in Colon Cancer by Suppression of Angiogenesis Signaling and Akt Activation

<b><i>Background/Aims:</i></b> Silencing of tumor suppressor genes (TSGs) and promotion of angiogenesis are associated with tumor development and metastasis. However, little is known if angiogenic molecules directly control TSGs and vice versa. <b><i>Methods:</i></b> A regulatory link between angiogenesis and down regulation of TSGs was evaluated using an anti-cancer agent, andrographolide (AGP) in cancer cells, mouse xenograft tissues and patient derived organoids through gene/protein expression, gene silencing, and immunohistochemical analyses. <b><i>Results:</i></b> AGP treatment demonstrated significant expression of RASSF1A and PTEN TSGs in colon cancer and other cancer cells, mouse tissues and organoids. Depletion of RASSF1A with siRNA limited cyclin D1 and BAX expression. SiRNA depletion of PTEN, upstream regulator of RASSF1A resulted in a 50% reduction in RASSF1A expression. Histopathological analysis of the AGP treated tumor sections showed significant reduction in vessel size, microvascular density and tumor mitotic index suggesting suppression of angiogenesis. This was corroborated by protein analysis demonstrating significant reductions in angiogenesis signaling pathway molecules VEGF₁₆₅, FOXM1, and pAkt, but significant elevation of the endogenous angiogenesis inhibitor Tsp-2. Treatment of cells with exogenous VEGF prevented the suppression of angiogenesis signaling by AGP, resulting in sustained expression of pAkt, an upstream down-regulator of RASSF1A. RASSF1A expression remained low in VEGF treated cells despite the addition of AGP. <b><i>Conclusion:</i></b> Our results demonstrate for the first time that AGP induces RASSF1A expression in colon cancer cells and is dependent on angiogenesis signaling events. Therefore, our research may facilitate novel therapeutic options for advanced colon cancer therapy

Histological assessment of the Sydney classification of endoscopic gastritis.

To determine the significance of the endoscopic classification of gastritis proposed by a working party at the World Congress of Gastroenterology in Sydney 1990, 167 patients undergoing upper alimentary endoscopy were prospectively assessed by comprehensive endoscopic and histological methods. Ninety eight patients had endoscopic mucosal changes of gastritis according to the Sydney classification. Twenty six (27%) of these had histologically normal biopsy specimens. This was not statistically significantly different to the 26 (38%) of 69 with normal endoscopies whose biopsy specimens were histologically normal (chi 2 = 1.857, p > 0.1). Forty three (62.5%) patients with normal endoscopies had histological gastritis. No histological counterpart was found for the macroscopic appearances of the gastric mucosa said to show inflammation proposed by the Sydney classification of gastritis. These findings confirm the inappropriateness of an endoscopic diagnosis of gastritis and it is suggested such a term should be reserved for the histological findings